O-Glucosylation of cis-Zeatin in Maize. Characterization of Genes, Enzymes, and Endogenous Cytokinins

Abstract

trans-Zeatin is a major and ubiquitous cytokinin in higher plants. cis-Zeatin has traditionally been viewed as an adjunct with low activity and rare occurrence. Recent reports of cis-zeatin and its derivatives as the predominant cytokinin components in some plant tissues may call for a different perspective on cis-isomers. The existence of a maize (Zea mays) gene (cisZOG1) encoding an O-glucosyltransferase specific to cis-zeatin (R.C. Martin, M.C. Mok, J.E. Habben, D.W.S. Proc Natl Acad Sci USA 98: 5922-5926) lends further support to this view. Results described here include the isolation of a second maize cisZOG gene, differential expression of cisZOG1 and cisZOG2, and identification of substantial amounts of cis-isomers in maize tissues. The open reading frame of cisZOG2 has 98.3% identity to cisZOG1 at the nucleotide level and 97.8% at the amino acid level. The upstream regions contain common and unique segments. The recombinant enzymes have similar properties, K m values of 46 and 96 m, respectively, for cis-zeatin and a pH optimum of 7.5. Other cytokinins, including N 6 -(⌬ 2 -isopentenyl)adenine, trans-zeatin, benzyladenine, kinetin, and thidiazuron inhibited the reaction. Expression of cisZOG1 was high in maize roots and kernels, whereas cisZOG2 expression was high in roots but low in kernels. cis-Zeatin, cis-zeatin riboside, and their O-glucosides were detected in all maize tissues, with immature kernels containing very high levels of the O-glucoside of cis-zeatin riboside. The results are a clear indication that O-glucosylation of cis-zeatin is a natural metabolic process in maize. Whether cis-zeatin serves as a precursor to the active trans-isomer or has any other unique function remains to be demonstrated.Cytokinins are plant hormones regulating cell division and a range of developmental events such as bud formation, leaf expansion, senescence, seed germination, and chloroplast formation (Mok, 1994). trans-Zeatin is a major and ubiquitous cytokinin in higher plants. Earlier cytokinin analyses detected ciszeatin and its derivatives in trace amounts in some plants, but due to their low activity (Schmitz et al., 1972), cis-isomers were viewed as adjunct to transisomers. Recent analyses, however, showed that the cis-isomers can be the dominant cytokinins at particular stages of development in plants such as chickpea (Cicer arietinum) and lupine (Lupinus albus; Emery et al., 1998Emery et al., , 2000. Moreover, the presence of cis-isomers was associated with male sterility in Mercurialis spp. flowers (Louis et al., 1990;Durand and Durand, 1994). These are indications that cis-isomers may have unique physiological functions. The ability to regulate the levels of cis-zeatin is evidenced by the maize (Zea mays) cisZOG1 gene, encoding an O-glucosyltransferase with specificity to cis-zeatin (Martin et al., 2001).O-Glucosylation is a major step in the metabolism of trans-zeatin ). The resulting O-glucosides seem to serve as storage compounds and are resistant to degradation by cytokinin oxidases (Armstrong, 1994...

N-Glucosylation of Cytokinins by Glycosyltransferases of Arabidopsis thaliana

“…Taken together with their low K m values toward these two substrates, it is clear that N 6 -benzyladenine and N 6 -isopentenyladenine are the preferred substrates for UGTs 76C1 and 76C2 from those tested. The K m values of UGTs 76C1 and 76C2 toward N 6 -benzyladenine, 0.09 and 0.04 mM, respectively, are similar to those reported in the study (28) of cis-ZOG1 and cis-ZOG2 toward cis-zeatin (0.046 and 0.096 mM, respectively). There are no kinetic data from other cytokinin-O-glycosyltransferases available for comparison.…”

“…In contrast to earlier studies on cytokinin O-glycosyltransferases from other plant species (27,28), recombinant UGTs from Arabidopsis recognized both trans-and cis-zeatin in vitro. Fig.…”

“…When these sequences are compared with the enzymes identified in Zea and Phaseolus by Mok and co-workers (24,25,27,28), only low sequence similarity was observed (Ͻ30%). The Zea and Phaseolus UGTs form a unique branch on the phylogenetic tree containing 107 Arabidopsis UGTs (Fig.…”

“…Although O-glucosylation and N-glucosylation of cytokinins have been described and reviewed, only O-glucosylation has been investigated in depth because of the availability of genes encoding the enzymes and biochemical analyses of their recombinant products (3). Genes encoding these enzymes from several plant species have been identified by Mok and coworkers (24,25,27,28), including ZOG1 from Phaseolus lunatus, ZOX1 from Phaseolus vulgaris, and cis-ZOG1 and cis-ZOG2 from maize. In contrast, nothing has been known of the N-glucosyltransferases of cytokinins, except early biochemical studies on partially purified enzyme preparations from a range of different plant species (29,30,35).…”

“…Conjugation of cytokinins also occurs and can involve Oxylosylation, O-glucosylation, and N-glucosylation (23)(24)(25)(26)(27)(28)(29)(30). Although all of these cytokinin glycosides are known to be inactive, their role in homeostasis is uncertain (3,13).…”